Accessibility of Telomeric Overhangs to Stabilizing Small-Molecule Ligands

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Human chromosomes have telomeric overhangs with repeating sequences that form G-quadruplexes (GQs).
• Small molecules stabilizing GQs can inhibit telomerase, a target for anti-cancer drugs.
• Understanding ligand accessibility to GQs within telomeric overhangs is crucial for drug development.

Purpose of the Study:

• To investigate how the architecture of telomeric overhangs influences the binding of small-molecule ligands to G-quadruplexes (GQs).
• To quantify the binding stoichiometry of a telomestatin derivative to telomeric overhangs of varying lengths and structures.
• To provide mechanistic insights for designing improved GQ-targeting anti-cancer agents.

Main Methods:

• Single-molecule fluorescence microscopy and stepwise photobleaching analysis to determine ligand binding stoichiometry.
• Synthesis and use of a fluorescently labeled oxazole telomestatin derivative (L1Cy5-7OTD).
• Ensemble fluorescence enhancement experiments with N-methyl mesoporphyrin IX (NMM) and computational modeling using a 1D lattice model.

Main Results:

• Longer telomeric overhangs accommodate more ligands but show lower-than-theoretical binding stoichiometry, saturating at six ligands.
• Increased ligand binding was observed with longer inter-G-quadruplex (GQ) spacers (9-nt vs. 3-nt).
• Modeling revealed positive cooperativity between GQ folding, negative cooperativity between ligands on opposing GQ faces, and reduced binding at double-stranded/single-stranded junctions.

Conclusions:

• Telomeric overhang architecture significantly dictates small-molecule accessibility to G-quadruplexes.
• Ligand binding is influenced by GQ folding cooperativity and spatial arrangement within the overhang.
• These findings offer mechanistic understanding to guide the rational design of GQ-targeting anticancer drugs.